Diverse Partners of the Partitioning ParB Protein in Pseudomonas aeruginosa

ABSTRACT In the majority of bacterial species, the tripartite ParAB-parS system, composed of an ATPase (ParA), a DNA-binding protein (ParB), and its target parS sequence(s), assists in the chromosome partitioning. ParB forms large nucleoprotein complexes at parS(s), located in the vicinity of origin of chromosomal replication (oriC), which after replication are subsequently positioned by ParA in cell poles. Remarkably, ParA and ParB participate not only in the chromosome segregation but through interactions with various cellular partners they are also involved in other cell cycle-related processes, in a species-specific manner. In this work, we characterized Pseudomonas aeruginosa ParB interactions with the cognate ParA, showing that the N-terminal motif of ParB is required for these interactions, and demonstrated that ParAB-parS-mediated rapid segregation of newly replicated ori domains prevented structural maintenance of chromosome (SMC)-mediated cohesion of sister chromosomes. Furthermore, using proteome-wide techniques, we have identified other ParB partners in P. aeruginosa, which encompass a number of proteins, including the nucleoid-associated proteins NdpA(PA3849) and NdpA2, MinE (PA3245) of Min system, and transcriptional regulators and various enzymes, e.g., CTP synthetase (PA3637). Among them are also NTPases PA4465, PA5028, PA3481, and FleN (PA1454), three of them displaying polar localization in bacterial cells. Overall, this work presents the spectrum of P. aeruginosa ParB partners and implicates the role of this protein in the cross-talk between chromosome segregation and other cellular processes. IMPORTANCE In Pseudomonas aeruginosa, a Gram-negative pathogen causing life-threatening infections in immunocompromised patients, the ParAB-parS system is involved in the precise separation of newly replicated bacterial chromosomes. In this work, we identified and characterized proteins interacting with partitioning protein ParB. We mapped the domain of interactions with its cognate ParA partner and showed that ParB–ParA interactions are crucial for the chromosome segregation and for proper SMC action on DNA. We also demonstrated ParB interactions with other DNA binding proteins, metabolic enzymes, and NTPases displaying polar localization in the cells. Overall, this study uncovers novel players cooperating with the chromosome partition system in P. aeruginosa, supporting its important regulatory role in the bacterial cell cycle.

"entangle" here is wrong/misleading. Entanglement often means DNA intertwines together randomly. SMC is known to prevent DNA entanglement, not promote it. I assume Kawalek et al mean "cohesion" instead of "entanglement", that SMC potentially coheses the 2 replicated sister chromosomes more when oriC-segregation by ParB-ParA is impaired. I would suggest using the word "cohese/cohesion" throughout the manuscript.
Minor comments: Line 72: PopZ is not an ATPase? Line 99 Replace "CTP processing" with CTP binding and hydrolysis? Line 302: add the word "putative" as in "we have identified putative proteins interacting with partitioning protein parB..." Reviewer #2 (Comments for the Author): ParAB-parS plays an important role in the faithful segregation of chromosomes. Kawalek et al have shown that impaired interaction of ParB and Par A proteins leads to growth retardation and increased frequency of anucleated cells. Further, smc deleted strains have been shown to reduce the detrimental effect of disruption of ParA-ParB interaction. Proteomics techniques have been used to identify the novel ParB interacting proteins that includes enzymes, nucleoid-associated proteins, and stress proteins etc. As such, the present study provides new insight into the role of ParA-ParB interaction in chromosome segregation and also identifies novel interacting partners of ParB. However, a triple deletion strain of parB, smc and mksB would have provided a deeper understanding of the genetic interaction of parABS with condensins. Overall, the manuscript is well written, and the experiments performed to support the conclusions. However, the authors should address the following points to strengthen their findings.
1. Bartosik et al 2004, have shown that ParB C-terminal domain interacts with ParA by yeast two hybrid assay; on the contrary, the present study shows ParB N-terminal interacts with ParA using BACTH system. Therefore, it may be required to validate the aforementioned interactions in-vitro or discuss this discrepancy in detail.
2. THE BACTH assay results suggest that point mutants of ParB do not interact with ParA. However, it is possible that point mutants still may interact with ParA, albeit with low affinity when compared to wild-type ParB. Is the BACTH assay sensitive enough to detect such weak interactions?
3.Do the point mutations in the N-terminal domain of ParB also affect its oligomeric state in solution and its DNA binding property? 4. smc deletion is known to cause defects in chromosome segregation, especially in rich media. Therefore, the effect of ParB in chromosome partitioning in smc null strain of P aeruginosa should also be studied in such conditions. 5.Authors can discuss how ParB interacting proteins would influence the ParA-ParB interaction or DNA binding activity of ParB.

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Comments for the authors:
ParAB-parS plays an important role in the faithful segregation of chromosomes. Kawalek et al have shown that impaired interaction of ParB and Par A proteins leads to growth retardation and increased frequency of anucleated cells. Further, smc deleted strains have been shown to reduce the detrimental effect of disruption of ParA-ParB interaction. Proteomics techniques have been used to identify the novel ParB interacting proteins that includes enzymes, nucleoid associated proteins, and stress proteins etc. As such, the present study provides new insight into the role of ParA-ParB interaction in chromosome segregation and also identifies novel interacting partners of ParB. However, a triple deletion strain of parB, smc and mksB would have provided deeper understanding on the genetic interaction of parABS with condensins. Overall, the manuscript is well written and the experiments performed support the conclusions. However, authors should address the following points to strengthen their findings.

Bartosik et al 2004, have shown that ParB C-terminal domain interacts with ParA by
yeast two hybrid assay, on the contrary the present study shows ParB N-terminal interacts with ParA using BACTH system. Therefore, it may be required to validate the aforementioned interactions in-vitro or discuss this discrepancy in detail.
2. THE BACTH assay results suggest that point mutants of ParB do not interact with ParA. However, it is possible that point mutants still may interact with ParA, albeit with low affinity when compared to wild-type ParB. Is the BACTH assay sensitive enough to detect such weak interactions?
3. Do the point mutations in the N-terminal domain of ParB also affect its oligomeric state in solution and its DNA binding property?
4. smc deletion is known to cause defects in chromosome segregation, especially in rich media. Therefore, the effect of ParB in chromosome partitioning in smc null strain of P aeruginosa should also be studied in such conditions. This work aimed for identification of the putative partners of Par proteins. We agree that the role of the most of them, (with the exception of the Min system although still not thoroughly investigated in P. aeruginosa), is unclear and awaits further studies focused on the particular partners. The significance of this study is in the demonstration of the wide spectrum of ParB partners, and hence processes within the cell, which can be linked with the process of DNA segregation.

Authors can discuss how ParB interacting proteins would influence the
Kawalek We attempted the construction of a double deletion strain ΔmksBEF ΔparAB by replacing parAB in an ΔmksBEF background with an antibiotic resistance cassette, however we repeatedly failed to obtain a viable mutant, so we could not proceed with the construction of the triple mutant. Perhaps less stringent conditions (temperature, concentration of antibiotic) are required for the selection of such strongly impaired mutant strain. Nevertheless we think that the above-mentioned studies explored this subject sufficiently, and showed that the MksBEF contribution to the segregation process becomes critical in the absence of the ParAB-parS system. It is worth to point out that in the current study we have used ΔmksBEF background to eliminate the 'backup' DNA segregation pathway, which we were hoping would allow us to see phenotypic defects of the constructed strains, if absence of the selected ATPases would have impaired functioning of the ParAB-parS system.

Overall, the manuscript is well written, and the experiments performed to support the conclusions.
However, the authors should address the following points to strengthen their findings.

1.Bartosik et al 2004, have shown that ParB C-terminal domain interacts with ParA by yeast two hybrid assay; on the contrary, the present study shows ParB N-terminal interacts with ParA using BACTH system. Therefore, it may be required to validate the aforementioned interactions in-vitro or discuss this discrepancy in detail.
The observation that ParB C-terminal domain interacts with ParA by yeast two hybrid (YTH) assay was puzzling at that time. Whereas self-association of ParB could be observed as strong interactions in YTH, the interactions between ParB and ParA in YTH were much weaker. At that time, the understanding of possible YTH system artifacts was not so advanced, and in the next years multiple sources of artifacts of the YTH data were summarized (e. We do consider such a possibility, as suggested previously in the discussion (lines 334-335). We have modified the results section to highlight this limitation of BACTH assay more clearly (lines 119-122). Nevertheless the final conclusion is that the single amino acids substitutions in the Nterminus of ParB have an impact on the ability to interact with ParA.

3.Do the point mutations in the N-terminal domain of ParB also affect its oligomeric state in solution and its DNA binding property?
We think that there is very limited (if any) influence of the analyzed ParB substitutions in the Nterminal part on the protein oligomerization and DNA (parS, half-parS) binding properties. This is based on the following observations (reported in the manuscript or previously published by us): 1) ParB G11A binding and spreading around parSs (and binding to half-parSs) was observed in ChIP-seq (ChiP-seq data available under accession number GSE213881) and no major effect of this amino acid substitution on the extent of ParB spreading (Fig. 2G).
2) N-terminally truncated ParB (Δ1-18) showed the same ability to silence the gene expression in the test plasmid as the full length ParB protein (Table 3, ref [45]). In the silencing test, overproduced ParB from one plasmid binds to parS on the compatible plasmid, spreads on DNA and forms a nucleoprotein complex leading to the reduced expression of genes required for plasmid replication. This is observed as a 1000-fold reduction in the efficiency of transformation with two plasmids. ParB variants with impaired DNA binding, dimerization or carrying substitutions in the N-terminal ParB domain (responsible for CTP binding an hydrolysis) do not demonstrate the gene silencing effect in this test (ref. [40], [54]). ParB variants with alanine substitutions at position 8 to 12 were analyzed with this test, and they did not differ from the WT ParB protein (data not shown).
3) Analyzed ParB variants formed foci when fused to YFP ( Fig. 2 and Figure S2).  (Petrushenko et al., 2011(Petrushenko et al., , doi: 10.1111(Petrushenko et al., /j.1365(Petrushenko et al., -2958(Petrushenko et al., .2011 or ~1% (Zhao et al., 2016, https://doi.org/10.1128. This indicates that there is no major influence of SMC on the segregation process in P. aeruginosa as judged by the analysis of anucleate cells content alone. In our hands in PAO1161 (a PAO1 derivative -discussion of the differences between the strains in ref [80]), the Δsmc strain cultures do not contain significantly more anucleate cells than WT when grown in rich LB medium at 37 o C, (doubling time 30±1 min) (Fig. 2E). The same is observed in M9 medium containing glucose (37 o C) or citrate (28 o C), conditions in which WT P. aeruginosa cultures have reduced doubling time (e.g. 110 min in case of medium with glucose) (Fig. S4AB). Of course, we have only looked at the amount of anucleate cells and there are other defects, which were recently described in an elegant study by Lioy et al.,(ref [48]), thus we did not analyze the phenotype of Δsmc mutant in detail as a part of this manuscript.

5.Authors can discuss how ParB interacting proteins would influence the ParA-ParB interaction or DNA binding activity of ParB.
The influence of partners on e.g. the extent of ParB spreading is indeed something to be investigated. Concomitantly, our data ( Fig 5A) indicate that, at least the four putative NTPases, are unlikely to compete with ParA for ParB binding, as they interact with truncated ParB 37-290 variant lacking the motif shown to be the ParA binding region. The molecular basis of the interaction between ParB and selected partners is currently investigated and will be included in another manuscript. The discussion has been modified to suggest the possible impact of partners on the indicated ParB properties (line 395). Dear Prof. Grazyna Jagura-Burdzy: Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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